Fibrin sealants are well known and extensively used in clinical settings to promote hemostasis, i.e., the prevention of blood loss through coagulation, the formation of a blood clot. Fibrin sealants are commonly used as adjuncts to conventional surgical techniques such as suturing and cautery to control bleeding when the aforementioned techniques are found to be ineffective or impractical. Fibrinogen and fibrin have been known to play a major role in wound healing and in blood coagulation for many years. Bergel, in 1909, used fibrin powder to seal the wall of a blood vessel (Schwartz S I, Shires G T, Spencer F C, et al. Principles of surgery 7th ed. McGraw Hill Co 1999; PP: 77-100). In cerebral surgery, fibrin was used for hemostasis as early as 1915 (Grey EG: Fibrin as a hemostatic in cerebral surgery. Surg Gynecol Obstet 1915; 21:452-4.). Young et al., have used fibrinogen to unite peripheral nerves (Young J Z, Medawar P B: Fibrin suture of peripheral nerves. Lancet 1940; 2:126). Tarlov and Bernard have also reported on the use of plasma clot to do the same (Tarlov IM, Bernard B: Plasma clot and silk suture of the nerves. Surg Gynecol Obstet 1943; 76:366.). However, in 1944 Cronkite et al. (CRONKITE LEP., LOZNER LEL., DEAVER CJM. Use of thrombin and fibrinogen in skin grafting. J. Amer. Med. Assoc., 1944, 124, 976-8.) were unsuccessful in the use of fibrinogen and thrombin in skin grafts in humans. According to Lerner and Binur (LERNER R., BINUR NS. Current status of surgical adhesives. J. Surg. Res., 1990, 48, 165-81), these and other studies were not successful, probably due to the use of low concentrations of fibrinogen. As a result, the use of this biological adhesive did not become popular until the 1970s.
Matras et al. in 1972, revived the interest in the use of fibrin for anastomosis of the nerves by increasing fibrinogen concentration (Matras H, Chiari F: Zur klebung von mikrogefassanastomosen, Proceedings, 13th Annual meeting, Dtsch Ges. F. Plast und wiederherstellungschirurgie. Thieme 1977; PP: 357). Kunderna in 1975, used autogenous cryoprecipitate to reconnect human digital nerves (Kunderna H, Matras H: Die Klinische anwendung der klebung von Nervananastomosen bei der Rekonstruktion verletzter peripherer Nervan. Wien Klin Wochenschr 1975, 87:495). Eventually, fibrin glue was found to be effective in sealing dural defects, splenic rupture and visceral organ tears. In 1976, Spangler and co-workers did the same in cardiac surgery (Spangler H P, Braun F: Die lokale anwendung von fibrinogen und kollagen zur blustillung der herzchirurgie. Wien Med Wochenschr 1976; 126:86). Fibrin glue was also found to be useful in enhancing bone repair, healing skin incisions, nerve grafting and for sealing mastoid and frontal cavities (Lerner R, Binur N S: Current research review-Current status of surgical adhesives. J Surg Res 1990; 48:165-81). Subsequently, fibrin glue has been widely used in nearly all surgical fields for hemostasis or tissue union (Blair S D, Backhouse C M, Harper R, et al: Comparison of absorbable materials for surgical haemostasis. Br J Surg 1988; 75: 969-71).
Early use of fibrin sealants had limited successes and sometimes frequent failures, primarily due to the methods of production from autologous human fibrinogen which produced inconsistent quality and quantity of fibrin sealant in the clinical setting. These early approaches relied solely on in-vivo concentrations of fibrinogen and thrombin, which produced varying levels of quality and quantity from each patient, resulting in a fibrin clot that was unpredictable. This improved slightly in 1944 when researchers mixed fibrinogen with bovine thrombin in an attempt to accelerate the formation of the fibrin clot. This was an improvement, but without the benefit of fractionation technology to purify and improve the concentration of the fibrinogen and fibrinogenic components, results were still not consistent.
The method of adding bovine thrombin however had its own drawbacks; that is, the chance of blood-borne transmission of transmissible diseases. During the early 1980s, wide acceptance of plasma products in the United States was significantly reduced due to the risk of contamination by the AIDS virus. The development of a commercial fibrin sealant product for the U.S. market became prohibitive, pending advances in pooled-plasma purification and viral-detection methodologies. Although several fibrin tissue sealants utilizing bovine thrombin were approved and clinically used in Europe for many years, the US Food and Drug Administration (FDA) only approved the first such product for use in the US in May, 1998 (FDA Product Approval Letter, Reference Numbers: 87-0508 and 87-0509).
During this period of advanced fibrin sealant development, various improvements were considered for the preparation and clinical applications of fibrin sealants. Hirsh et al., U.S. Pat. No. 5,643,192 describes autologous fibrin glue including a fibrinogen component and a thrombin component, both prepared from a single donor plasma source. Hirsh, however, does not envision utilizing anything other than the donor's own thrombin as the clotting agent. MacPhee et al. U.S. Pat. No. 6,117,425 describes the supplementation of fibrin sealant with one or more compositions whereby the fibrinogen forms a fibrin matrix when in the presence of thrombin, Ca++ and water and allows the added composition to be released from the fibrin matrix over a sustained period that is greater than the period obtained according to simple diffusion kinetics. Similarly, Marx U.S. Pat. No. 5,651,982 describes supplemented fibrin glue produced with either autologous or bovine thrombin and with liposomes embedded in the glue after clotting. The liposomes embedded within the fibrin glue contain biologically active ingredients that would be released over an extended period of time within the body. However, Marx describes a fibrin sealant where the liposomes are embedded only after the coagulation has occurred involving a clotting cascade generated with thrombin and fails to envision utilizing a fibrin sealant that is produced with anything other than thrombin as a clotting agent for the fibrin.
In 1993 researchers from the Center for the Study of Venoms and Venomous Animals—CEVAP of Sāo Paulo State University—UNESP standardized and reported on another type of fibrin glue derived from snake venom (luan, F C., Thomazini I A., Mendes-Giannini M J S., Toscano E., Viterbo F., Moraes R A., Barraviera B., Cola de fibrina derivada de veneno de cobra e processo para sua preparacão. Patente requerida junto ao Instituto Nacional de Propriedade Industrial. Rev. Propried. Industr., 1993, 1165, 27: Viterbo F., Thomazini IA., Giannini M J S M., Reparacão de nervos periféricos corn cola de fibrina derivada de veneno de cobra. Resultados preliminares. Acta Cir. Bras., 1993, 8, 85). This new glue consists of a cryoprecipitate containing human, bovine, bubaline or equine fibrinogen and a “thrombin-like” fraction of snake venom. This glue is different from the traditional glues since it uses animal instead of human fibrinogen and it also substitutes bovine thrombin for a “thrombin-like” enzyme extracted from Crotalus durissus teffificus venom which was isolated by Raw et al. (Raw I., Rocha M C., Esteves M I., Kamiguti A S., Isolation and characterization of a thrombin-like enzyme from the venom of Crotalus durissus terrificus. Braz. J. Med. Biol. Res., 1986, 19, 333). The “thrombin-like” enzyme is much more potent than bovine thrombin in the conversion of fibrinogen into fibrin (Thomazini-Santos I A., Avaliacão do tempo de coagulacão da trombina bovina, da reptilase e da fracão do “tipo-trombina” de serpentes Crotalus durissus teffificus, empregando-se crioprecipitado de diferentes espécies animais. Botucatu: Universidade Estadual Paulista, Faculdade de Medicina, 1996. 71 p). The utilization of this glue has the advantage of permitting the use of homologous, autologous and heterologous fibrinogen, according to necessity. Applicability of fibrin glue derived from snake venom was subsequently tested and approved for use in different animal tissues.
Viterbo et al. and luan et al., (luan F C., Thomazini I A., Mendes-Giannini M S., Viterbo F., Toscano E., Moraes R A., Barraviera B.; Reparation of peripheral nerves with fibrin glue prepared from snake venom. Preliminary results. Rev. Paul. Med., 1995, 113, 1000-2) tested the fibrin glue derived from snake venom in the repair of sciatic nerve of Wistar rats and observed its good hemostatic and adhesive properties, as well as a satisfactory regeneration of the sealed nerves. Viterbo et al. used bubaline, equine, bovine and human fibrinogen. These authors concluded that fibrin glue derived from snake venom and bubaline fibrinogen showed to be a major alternative for the repair of peripheral nerves.
Leite et al. (Leite C V S., Naresse L E., Saad L H C., Thomazini I A., Barraviera B., Kobayasi S.; Cicatrizacão intestinal—efeito da cola de fibrina derivada de veneno de cobra na anastomose do cólon de ratos. In: Congresso Brasileiro De Colo-Proctologia, 43, Recife, 1994. Anais . . . Recife, 1994) tested the fibrin glue derived from snake venom in anastomoses of colon of Wistar rats with good results. The glue they used was made up of snake venom fraction and bubaline fibrinogen.
Stolf et al. (Stolf H O., Barraviera S R C S., Thomazini I A., Giannini M J S M., Toscano E., Barraviera B.; Cola de fibrina derivada de veneno de cobra. Uso experimental em cirurgia dermatológica. In: Congresso Da Sociedade Brasileira De Dermatologia, 48, Curitiba, 1993. Anais . . . Curitiba, 1993) evaluated this fibrin glue in skin of rabbits subjected to elliptical skin incision on the trunk. The results obtained suggested that the glue acts efficiently on the healing process due to an increase of a fibrin network.
Subsequently, Edwardson et al., U.S. Pat. No. 5,770,194 described an autologous fibrin sealant composition produced with a thrombin-like enzyme derived from snake venom (Batroxobin) and without the use of any bovine derived additives such as Aprotinin, derived from bovine lung, or thrombin which could carry blood-transmitted infections, infectious agents and viruses pathogenic to mammals. Bovine thrombin has been known to carry the infectious agent bovine spongiform encephalitis (BSE) and other viruses. Furthermore, bovine thrombin is a potent antigen, which can cause immunological reactions in humans. Thus, the use of bovine thrombin could result in the recipient of the bovine thrombin being adversely affected. Additionally, Edwardson et al., U.S. Pat. No. 5,770,194 described a kit comprising autologous fibrin monomer or non-crosslinked fibrin which can be polymerized to form fibrin sealant utilizing a thrombin-like enzyme derived from snake venom thus avoiding concerns for blood-transmitted infections and or viruses that are pathogenic to mammals. The present inventors have observed that Edwardson et al. do not envision the use of supplemented autologous tissue sealants where there is the added benefit of a therapeutic composition which is to be released from the fibrin matrix over a sustained period that is greater than the period which might normally be obtained through standard diffusion kinetics. Additionally, the degradation or resorption rate of fibrin sealants produced from thrombin-like enzymes may be tailored to have different diffusion or resorption rates than fibrin sealants produced with thrombin.